The present invention is generally related to fluorescent molecules for cellular imaging applications and, in particular, to photo-caged fluorescent molecules which are cell permeable and useful for the study of cellular imaging applications and tracing molecular transfer in cellular gap junctions.
Photo-caged fluorescent dyes have wide applications in tracking the spatiotemporal dynamics of molecular movements in biological systems. These caged tracers are weakly or non-fluorescent when key functional groups of fluorophores are masked by photo-labile protecting groups, or cages. Photo-activation “uncages” the molecule by removing the protecting group and abruptly switches on the fluorescence of the parent dyes. By selectively photoactivating labeled molecules present in a certain area of a cell, the progression of the fluorescent signal can then be tracked over time as it moves away from the “uncaging” site into the dark surrounding area. (Adams, et al., 1993; Politz, 1999; Mitchison, et al., 1998).
Caged fluorochromes are covalently linked to a macromolecule and then, once inside the cell, the caging moiety is usually removed by a pulse of long-wavelength ultraviolet (“UV”) light ranging from about 330 nm to 390 nm. Caging groups are typically designed to be released by long-wavelength UV light rather than short-wavelength UV light because long-wavelength UV light is less harmful to cells. To minimize cell damage, the use of a minimal amount of UV illumination is desirable. At the same time, an essential requirement of a caged fluorophore is that its fluorescence excitation wavelength does not overlap with the wavelength of UV light used for uncaging.
Macromolecules labeled with caged fluorochromes are usually microinjected into the cell because the macromolecules are impermeable to the cell membrane. Small caged fluorescent molecules that are cell permeable or can be loaded into cells to high concentrations have not been described yet. However, some oligonucleotides labeled with caged fluorochromes can be taken up by cultured cells alone or after being complexed with cationic lipids. (Politz, 1999).
U.S. Pat. No. 5,830,912 to Gee et al. describes a class of fluorescent dyes derived from 6,8-difluoro-7-hydroxycoumarin, some of which may be photo-caged. However, the excitation maximum of most of the compounds related to 6,8-difluoro-7-hydroxycoumarin is at about 370 nm, which overlaps with the wavelengths of UV light typically used for uncaging. A few compounds described herein can be excited above 400 nm, but the uncaging cross sections of these compounds have not been described, neither the cell permeability has been described. Thus, these molecules are not ideally suited for use as photo-caged fluorescent molecules.
U.S. Pat. No. 5,635,608 to Haugland et al. describes caged compounds with a photoremoveable α-carboxy-substituted o-nitrobenzyl group. Except for the disclosure of one rhodamine derivative, the disclosed compounds are not fluorophores. Rather, they are biomolecules with a caging group directly attached, so that photoactivation restores the bioactivity of the molecule but does not result in fluorescence.
Fluorescent probes and tracers used in cellular imaging may be used to study the transfer of molecules through gap junctions in cells. Malfunctions of intercellular communications through connexin channels and gap junctions are associated with diseases such as deafness, peripheral neurophathy, cataracts, hereditary malfunctions of the cardiovascular system, and Chagas' disease. Thus, it is of pharmaceutical interest to develop high throughput screening technology for isolating specific modulators of gap junctions.
Several techniques exist for studying gap junction transfers. Microinjection of membrane-impermeant tracers such as Lucifer Yellow or neurobiotin is a classical technique for assaying molecular permeability of connexin channels (Meda, 2001). It allows for selective loading of tracers into cells of choice. However, the method is associated with a number of major limitations. First, cell membranes are usually damaged during the operation, so special skill and extra care are needed to minimize cell injury. Also, only a limited number of cells can be injected at a time, so it is not convenient to study intercellular communications in cell populations or tissue preparations. Finally, the microinjection process may disrupt the extracellular and intracellular concentrations of other molecules affecting gap junction permeability. For example, extracellular calcium ion (“Ca2+”) concentrations are typically greater than 1 mM, which is more than 104 times higher than intracellular Ca2+ concentrations, which are typically less than 0.1 μM at the resting state. It is very difficult to maintain the intracellular Ca2+ concentration at a normal physiological level during microinjection. Thus, studies of fluctuations in concentration of molecules such as Ca2+ in relation to gap junction permeability may be adversely affected. The technique known as “scrape loading” suffers from similar drawbacks (el-Fouly, et al., 1987).
An additional method, known as the dual whole-cell patch clamp method, has also been applied to determine kinetics and conductance of gap junctions in both primary cultures and cell lines expressing exogenous connexins (Van Rijen, et al., 2001). This method has the known advantages of the whole-cell patch clamp method, which uses fewer electrodes and generates more stable recordings. However, the accuracy of junctional current measurement is affected by the relative values of uncompensated electrode resistances, nonjunctional resistances, junctional resistance and seal resistances (Harris, 2001). Thus, caution must be taken in order to maintain an accurate measurement. This method has also been used for dye-passage experiments due to the low diffusional barrier of the patch pipette. While the technique offers some abilities to alter cytoplasmic constituents, it can also cause problems when concentrations of cellular ions, such as Ca2+, must be accurately controlled, or when certain important cytoplasmic factors should be prevented from being diluted out in the pipette.
Fluorescence Recovery After Photobleaching (“FRAP”) is another technique for tracking molecular movements in cells (Deleze, et al., 2001). At the present time, the laser power of most commercially available confocal systems may be adequate for spot photobleaching, but generally much higher laser power is required for rapid photobleaching of whole cells as required by the gap junction FRAP (“GJ-FRAP”) technique. This intense laser illumination may damage cells. Moreover, the GJ-FRAP technique maybe incompatible with multi-color imaging when other biochemical changes inside cells need to be studied by fluorescent sensors.
Ideally, caged fluorophores used in cellular imaging applications should be efficiently photoactivated at low levels of UV radiation, be capable of localized uncaging, have robust fluorescence enhancement after uncaging, and have flexible chemistry for bioconjugation and cellular delivery. They should also be cell permeable, particularly for the study of intercellular communications, and compatible with other imaging techniques to allow simultaneous studies.